In FY15 we published the first intent-to-treat clinical trial of anti-CD19 chimeric antigen receptor based therapy for children and young adults with relapsed/refractory B cell malignancies (12C0112, NCT01593696). Twenty-one patients were enrolled and this trial observed a very high response rate in this patient population. The major toxicities were related to cytokine release syndrome and were reversible with immunosuppression. We observed that efficacy is correlated with several biomarkers including expansion of the CD19-CAR expressing T cells. Through this experience we have also focused on improving the grading system and treatment algorithm for cytokine release syndrome. We developed a consensus manuscript with collaborators for four institutions to accomplish this and published this manuscript in BLOOD in 2014. In FY15, we published a manuscript detailing experience treating patients with activated NK cells administered following allogeneic stem cell transplantation for high risk pediatric cancer. This built upon a previous published study of allogeneic stem cell transplantation for patients with high-risk pediatric sarcomas, that used a non-myeloablative allogeneic peripheral blood stem cell transplant for patients with matched sibling donors to treat patients with ultra-high risk pediatric solid tumors. Results showed a high rate of GVHD that was unexpected given that the grafts were T cell depleted and NK cells have not previously been implicated in causing GVHD. These results were published in Blood in 2014. The clinical trial has subsequently been modified to incorporate GVHD prophylaxis. In FY15, we submitted a manuscript detailing results of the only clinical trial to date of checkpoint blockade in children with cancer. We observed disease stabilization on this study, but no objective antitumor responses. We also have demonstrated that children experience autoimmune toxicity with this agent, which is similar in nature and severity to that observed in adults. In FY15, we completed a manuscript detailing treatment of patients treated on our combined tumor vaccine, immune reconstitution trial (NCI 07-c-0206). The biologic endpoints on this study regarding immune reconstitution are discussed in Project I, but we also observed a favorably 5-year overall survival rate for patients with metastatic Ewing sarcoma enrolled after completion of frontline therapy. These results are intent-to-treat and are significantly better than observed on our previous study of immune reconstitution/dendritic cell vaccine at the National Cancer Institute. This study provides an entirely novel approach to consolidating remissions in patients with high-risk sarcomas and raises a larger question regarding the potential role for cytotoxic induced lymphopenia in increasing the risk of cancer recurrence. In FY15, we continued enrollment on a trial using a genetically engineered T cell receptor targeting NY-ESO-1+ in HLA-A2+ patients with synovial sarcoma. This trial seeks to reproduce promising results seen in a trial administering similar T cells with high dose IL2 to patients with sarcoma and melanoma and represents a collaboration with Adaptimmune. We have observed a striking 60% objective response rate in these aggressive sarcomas. Notably, this trial did not utilize interleukin-2 and thus avoids substantial toxicity associated with this agent. Given the dismal outcomes for patients with metastatic synovial sarcoma, we believe that this agent has a high likelihood of FDA approval and are working with Adaptimmune to craft a registration study which could serve as the basis for accelerated approval for this orphan disease. In FY15 we continued enrollment on a trial using autologous activated NK cells for the treatment of solid tumors incorporating human interleukin-15 following infusion of activated NK cells. This represents the first use of recombinant human interleukin-15 in children. We observed significant toxicity with this agent in one patient and thus far do not have definitive data regarding whether the agent augments the expansion, persistence or effectiveness of activated NK cell infusions. In FY15 we had brisk enrollment of patients on a novel clinical trial using a chimeric antigen receptor targeting GD2, which incorporates both a CD28 and an OX40 costimulatory domain. Because of the potential for neurotoxicity with this agent due to low level expression of GD2 on peripheral nerves, the trial began with a very low starting dose and used a cautious dose escalation schema. We have observed no evidence for neurotoxicity or other autoimmune toxicity. We are currently enrolling patients at a dose level of 3 x 106 cells/kg and see very significant expansion in vivo. Notably, these cells are not persistent which is consistent with our discovery that CD28 costimulation diminishes persistence in highly activated CAR T cells.